Initiation is the most complex, tightly regulated stage of eukaryotic protein synthesis. The process begins with formation of the 48S initiation complex (48S IC) at the initiation codon of mRNA. First, the 43S preinitiation complex (43S PIC) comprising the 40S ribosomal subunit, the eIF2?GTP?Met-tRNAMeti ternary complex and eukaryotic initiation factors eIF3, eIF1 and eIF1A binds to the cap-proximal region of mRNA in a step that is mediated by eIFs 4A, 4B and 4F, which cooperatively unwind the cap-proximal region, allowing for 43S PIC association. The 43S PIC then scans downstream to the initiation codon where it forms the 48S IC with the established codon-anticodon interaction. Scanning on structured mRNAs additionally requires the DExH-box protein DHX29 that binds directly to 40S subunits. eIFs 1 and 1A play key roles in ensuring the fidelity of initiation codon selection. Initiation codon recognition triggers dissociation of eIF1, eIF5-induced hydrolysis of eIF2-bound GTP and release of Pi. Subsequent joining of a 60S subunit is promoted by the translational GTPase eIF5B. Initiation on some viral mRNAs is mediated by an internal ribosome entry site (IRES). IRESs are highly structured RNA elements that promote 5?-end independent recruitment of the 40S subunit via non-canonical interactions with the 40S subunits and/or eIFs. Dysregulation of translation initiation is frequently observed in devastating diseases and is therefore becoming a focus for chemo-therapeutic intervention. Although the factors required for initiation have been identified, and their principal roles determined, important details concerning its molecular mechanism, regulation and alternative modes remain unknown. Characterization of these details is therefore a priority. We have reconstituted the entire translation cycle in vitro, which gives us the unique opportunity to address critical gaps in understanding of the mechanisms of mammalian initiation and the regulation of translation using biochemical and complementary biophysical and cell biology approaches. Aim 1 will concern characterization of the mechanisms by which DHX29 promotes scanning, eIF5B stabilizes Met-tRNAiMet on the 40S subunit and both factors influence initiation codon selection. In Aim 2, we will focus on investigating the mechanisms of physiologically important initiation with Leu-tRNALeu at CUG codons, and on non-AUG triplets during repeat-associated non-AUG (RAN) translation, which occurs on expansion repeats in mRNAs transcribed from genes that are responsible for severe neurodegenerative diseases. Aim 3 is devoted to elucidation of the molecular mechanism of initiation on the IRES located in the 5'UTR of Cricket paralysis virus RNA, which has a unique structure and that our preliminary data suggest can use novel mechanisms for initiation. Aim 4 concerns the cellular function and mechanism of action of Schlafen14, a novel endoribonuclease that binds 80S ribosomes and cleaves rRNA and ribosome-bound mRNAs. It is thus implicated in translational control, and likely influences this process in a previously undescribed manner.